Lipoarabinomannan modification as a source of phenotypic heterogeneity in host-adapted Mycobacterium abscessus isolates.

Mycobacterium abscessus is increasingly recognized as the causative agent of chronic pulmonary infections in humans. One of the genes found to be under strong evolutionary pressure during adaptation of M. abscessus to the human lung is embC which encodes an arabinosyltransferase required for the biosynthesis of the cell envelope lipoglycan, lipoarabinomannan (LAM). To assess the impact of patient-derived embC mutations on the physiology and virulence of M. abscessus, mutations were introduced in the isogenic background of M. abscessus ATCC 19977 and the resulting strains probed for phenotypic changes in a variety of in vitro and host cell-based assays relevant to infection. We show that patient-derived mutational variations in EmbC result in an unexpectedly large number of changes in the physiology of M. abscessus, and its interactions with innate immune cells. Not only did the mutants produce previously unknown forms of LAM with a truncated arabinan domain and 3-linked oligomannoside chains, they also displayed significantly altered cording, sliding motility, and biofilm-forming capacities. The mutants further differed from wild-type M. abscessus in their ability to replicate and induce inflammatory responses in human monocyte-derived macrophages and epithelial cells. The fact that different embC mutations were associated with distinct physiologic and pathogenic outcomes indicates that structural alterations in LAM caused by nonsynonymous nucleotide polymorphisms in embC may be a rapid, one-step, way for M. abscessus to generate broad-spectrum diversity beneficial to survival within the heterogeneous and constantly evolving environment of the infected human airway.

Role of succinyl substituents in the mannose-capping of lipoarabinomannan and control of inflammation in Mycobacterium tuberculosis infection.

The covalent modification of bacterial (lipo)polysaccharides with discrete substituents may impact their biosynthesis, export and/or biological activity. Whether mycobacteria use a similar strategy to control the biogenesis of its cell envelope polysaccharides and modulate their interaction with the host during infection is unknown despite the report of a number of tailoring substituents modifying the structure of these glycans. Here, we show that discrete succinyl substituents strategically positioned on Mycobacterium tuberculosis (Mtb) lipoarabinomannan govern the mannose-capping of this lipoglycan and, thus, much of the biological activity of the entire molecule. We further show that the absence of succinyl substituents on the two main cell envelope glycans of Mtb, arabinogalactan and lipoarabinomannan, leads to a significant increase of pro-inflammatory cytokines and chemokines in infected murine and human macrophages. Collectively, our results validate polysaccharide succinylation as a critical mechanism by which Mtb controls inflammation.

Intrapulmonary Treatment with Mycobacteriophage LysB Rapidly Reduces Mycobacterium abscessus Burden.

Intrinsic and acquired antibiotic resistance in Mycobacterium abscessus presents challenges in infection control, and new therapeutic strategies are needed. Bacteriophage therapy shows promise, but variabilities in M. abscessus phage susceptibility limits its broader utility. We show here that a mycobacteriophage-encoded lysin B (LysB) efficiently and rapidly kills both smooth- and rough-colony morphotype M. abscessus strains and reduces the pulmonary bacterial load in mice. LysB aerosolization presents a plausible treatment for pulmonary M. abscessus infections.

Mycobacterium leprae Infection in a Wild Nine-Banded Armadillo, Nuevo León, Mexico.

Nine-banded armadillos (Dasypus novemcinctus) are naturally infected with Mycobacterium leprae and are implicated in the zoonotic transmission of leprosy in the United States. In Mexico, the existence of such a reservoir remains to be characterized. We describe a wild armadillo infected by M. leprae in the state of Nuevo León, Mexico.

Respirable Clofazimine Particles Produced by Air Jet Milling Technique Are Efficacious in Treatment of BALB/c Mice with Chronic Mycobacterium tuberculosis Infection.

Tuberculosis (TB) remains a major cause of morbidity and mortality, particularly in low- and middle-income countries where access to health care workers, cold-chain storage, and sterile water sources may be limited. Inhaled drug delivery is a promising alternative to systemic delivery of antimycobacterial drugs, as it enables rapid achievement of high infection-site drug concentrations. The off-patent drug clofazimine (CFZ) may be particularly suitable for this route, given its known systemic toxicities. In this study, micronized CFZ particles produced by air jet milling were assessed for shelf-stability, pharmacokinetics, and anti-TB efficacy by the oral and pulmonary routes in BALB/c mice. Intratracheal instillation of micronized CFZ particles produced several-fold higher lung concentrations after a single 30 mg/kg dose compared to delivery via oral gavage, and faster onset of bactericidal activity was observed in lungs of mice with chronic Mycobacterium tuberculosis infection compared to the oral route. Both infection status and administration route affected the multidose pharmacokinetics (PK) of micronized CFZ. Increased lung and spleen accumulation of the drug after pulmonary administration was noted in infected mice compared to naive mice, while the opposite trend was noted in the oral dosing groups. The infection-dependent PK of inhaled micronized CFZ may point to a role of macrophage trafficking in drug distribution, given the intracellular-targeting nature of the formulation. Lastly, air jet milled CFZ exhibited robustness to storage-induced chemical degradation and changes in aerosol performance, thereby indicating the suitability of the formulation for treatment of TB in regions with limited cold chain supply.

Inhaled tigecycline is effective against Mycobacterium abscessus in vitro and in vivo.

BACKGROUND: Mycobacterium abscessus causes chronic pulmonary infections. Owing to its resistance to most classes of antibiotics, treatment is complex and cure rates are only 45%. Tigecycline is active against M. abscessus, but severe toxicity and the need for IV administration limit its use. OBJECTIVES: To assess the potential of inhaled tigecycline as a treatment for M. abscessus pulmonary disease, by measuring its efficacy in a mouse model of chronic M. abscessus pulmonary disease, establishing the intracellular activity of tigecycline against M. abscessus in human macrophages and measuring the activity of tigecycline in the sputum of cystic fibrosis patients. METHODS: We infected GM-CSF knockout mice with M. abscessus by intrapulmonary aerosol. Infected mice were treated with tigecycline in 0.25, 1.25 and 2.5 mg doses, by inhalation, or untreated, for 28 days. Tigecycline was added to human peripheral blood-derived macrophages infected with M. abscessus to assess its intracellular activity. We performed a time-kill kinetics experiment of tigecycline against M. abscessus with and without sputum of cystic fibrosis patients. RESULTS: Inhaled tigecycline proved highly effective against M. abscessus in GM-CSF knockout mice. The effect was dose dependent. Tigecycline showed potent activity against M. abscessus in macrophages and retained most of its activity in the presence of sputum of cystic fibrosis patients. CONCLUSIONS: Inhaled tigecycline may represent a viable treatment option for M. abscessus pulmonary disease, where treatment outcomes are currently very poor. A stable and safe formulation is required to proceed to further pharmacodynamic studies and ultimately clinical trials.

Development and Characterization of a Dry Powder Formulation for Anti-Tuberculosis Drug Spectinamide 1599.

PURPOSE: Human tuberculosis (TB) is a global health problem that causes nearly 2 million deaths per year. Anti-TB therapy exists, but it needs to be administered as a cocktail of antibiotics for six months. This lengthy therapy results in low patient compliance and is the main reason attributable to the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis. METHODS: One alternative approach is to combine anti-TB multidrug therapy with inhalational TB therapy. The aim of this work was to develop and characterize dry powder formulations of spectinamide 1599 and ensure in vitro and in vivo delivered dose reproducibility using custom dosators. RESULTS: Amorphous dry powders of spectinamide 1599 were successfully spray dried with mass median aerodynamic diameter (MMAD) = 2.32 ± 0.05 µm. The addition of L-leucine resulted in minor changes to the MMAD (1.69 ± 0.35 µm) but significantly improved the inhalable portion of spectinamide 1599 while maintaining amorphous qualities. Additionally, we were able to demonstrate reproducibility of dry powder administration in vitro and in vivo in mice. CONCLUSIONS: The corresponding systemic drug exposure data indicates dose-dependent exposure in vivo in mice after dry powder intrapulmonary aerosol delivery in the dose range 15.4 - 32.8 mg/kg.

Yersinia pestis Survival and Replication in Potential Ameba Reservoir.

Plague ecology is characterized by sporadic epizootics, then periods of dormancy. Building evidence suggests environmentally ubiquitous amebae act as feral macrophages and hosts to many intracellular pathogens. We conducted environmental genetic surveys and laboratory co-culture infection experiments to assess whether plague bacteria were resistant to digestion by 5 environmental ameba species. First, we demonstrated that Yersinia pestis is resistant or transiently resistant to various ameba species. Second, we showed that Y. pestis survives and replicates intracellularly within Dictyostelium discoideum amebae for ˃48 hours postinfection, whereas control bacteria were destroyed in <1 hour. Finally, we found that Y. pestis resides within ameba structures synonymous with those found in infected human macrophages, for which Y. pestis is a competent pathogen. Evidence supporting amebae as potential plague reservoirs stresses the importance of recognizing pathogen-harboring amebae as threats to public health, agriculture, conservation, and biodefense.

Mycobacterium bovis hosted by free-living-amoebae permits their long-term persistence survival outside of host mammalian cells and remain capable of transmitting disease to mice.

Bovine tuberculosis (TB) is a zoonotic disease caused by Mycobacterium bovis. Despite intensive TB control campaigns, there are sporadic outbreaks of bovine TB in regions declared TB free. It is unclear how M. bovis is able to survive in the environment for long periods of time. We hypothesized that Free-living amoebae (FLA), as ubiquitous inhabitants of soil and water, may act as long-term reservoirs of M. bovis in the environment. In our model, M. bovis would be taken up by amoebal trophozoites, which are the actively feeding, replicating and mobile form of FLA. Upon exposure to hostile environmental conditions, infected FLA will encyst and provide an intracellular niche allowing their M. bovis cargo to persist for extended periods of time. Here, we show that five FLA species (Acanthamoeba polyphaga, Acanthamoeba castellanii, Acanthamoeba lenticulata, Vermamoeba vermiformis and Dictyostellium discoideum) are permissive to M. bovis infection and that the M. bovis bacilli may survive within the cysts of four of these species for over 60 days. We further show that exposure of M. bovis-infected trophozoites and cysts to Balb/c mice leads to pulmonary TB. This work describes for the first time that FLA carrying M. bovis can transmit TB.

Therapeutic Potential of the Mycobacterium tuberculosis Mycolic Acid Transporter, MmpL3.

In recent years, whole-cell-based screens for novel small molecule inhibitors active against Mycobacterium tuberculosis in culture followed by the whole-genome sequencing of spontaneous resistant mutants have identified multiple chemical scaffolds thought to kill the bacterium through the inactivation of the mycolic acid transporter, MmpL3. Consistent with the fact that MmpL3 is required for the formation of the mycobacterial outer membrane, we have conclusively shown in this study, using conditionally regulated knockdown mutants, that mmpL3 is required for the replication and viability of M. tuberculosis, both under standard laboratory growth conditions and during the acute and chronic phases of infection in mice. Speaking for the vulnerability of this target, silencing mmpL3 had a rapid bactericidal effect on actively replicating cells in vitro and reduced by 3 to 5 logs in less than 4 weeks the bacterial loads of acutely and chronically infected mouse lungs, respectively. Depletion of MmpL3 further rendered M. tuberculosis hypersusceptible to MmpL3 inhibitors. The exquisite vulnerability of MmpL3 at all stages of the infection establishes this transporter as an attractive new target with the potential to improve and shorten current drug-susceptible and drug-resistant tuberculosis chemotherapies.

Exposure to a cutinase-like serine esterase triggers rapid lysis of multiple mycobacterial species.

Mycobacteria are shaped by a thick envelope made of an array of uniquely structured lipids and polysaccharides. However, the spatial organization of these molecules remains unclear. Here, we show that exposure to an esterase from Mycobacterium smegmatis (Msmeg_1529), hydrolyzing the ester linkage of trehalose dimycolate in vitro, triggers rapid and efficient lysis of Mycobacterium tuberculosis, Mycobacterium bovis BCG, and Mycobacterium marinum. Exposure to the esterase immediately releases free mycolic acids, while concomitantly depleting trehalose mycolates. Moreover, lysis could be competitively inhibited by an excess of purified trehalose dimycolate and was abolished by a S124A mutation affecting the catalytic activity of the esterase. These findings are consistent with an indispensable structural role of trehalose mycolates in the architectural design of the exposed surface of the mycobacterial envelope. Importantly, we also demonstrate that the esterase-mediated rapid lysis of M. tuberculosis significantly improves its detection in paucibacillary samples.

GM-CSF knockout mice for preclinical testing of agents with antimicrobial activity against Mycobacterium abscessus.

OBJECTIVES: Of the non-tuberculous mycobacteria, Mycobacterium abscessus is particularly refractory to antimicrobial therapy and new agents with activity against these pathogens are urgently needed. The screening of candidate antimicrobial agents against M. abscessus requires a relevant and reproducible animal model of chronic infection. Granulocyte-macrophage colony-stimulating factor knockout (GM-CSF KO) mice were used to develop a new animal model of chronic pulmonary M. abscessus infection that can be used for preclinical efficacy testing of antimicrobial drugs. METHODS: GM-CSF KO mice were infected with a clinical isolate of M. abscessus via intrapulmonary aerosol delivery using a microsprayer device. The clinical condition, histology and cfu of M. abscessus-infected GM-CSF KO mice were evaluated over a period of 4 months. Mice were treated with azithromycin (100 mg/kg) by oral gavage and the clinical condition, histology and bacterial burden was determined after 2 weeks of treatment. RESULTS: We show that pulmonary infection of GM-CSF KO mice with M. abscessus results in a chronic pulmonary infection that lends itself to preclinical testing of new antimicrobial drugs against this bacterium. Azithromycin treatment of M. abscessus-infected GM-CSF KO mice resulted in a lower bacterial burden in the lungs and spleen, weight gain and significant improvement in lung pathology. CONCLUSIONS: Intrapulmonary aerosol infection of GM-CSF KO mice with M. abscessus is a useful animal model for studying pathogenesis as well as pre-clinical testing of new compounds against M. abscessus in acute or chronic phases of infection.

Microhemorrhage is an early event in the pulmonary fibrotic disease of PECAM-1 deficient FVB/n mice.

Platelet Endothelial Cell Adhesion Molecule 1 (PECAM-1) deficient mice in the FVB/n strain exhibit fatal chronic pulmonary fibrotic disease. The illness occurs in the absence of a detectable pro-inflammatory event. PECAM-1 is vital to the stability of vascular permeability, leukocyte extravasation, clotting of platelets, and clearance of apoptotic cells. We show here that the spontaneous development of fibrotic disease in PECAM-1 deficient FVB/n mice is characterized by early loss of vascular integrity in pulmonary capillaries, resulting in spontaneous microbleeds. Hemosiderin-positive macrophages were found in interstitial spaces and bronchoalveolar lavage (BAL) fluid in relatively healthy animals. We also observed a gradually increasing presence of hemosiderin-positive macrophages and fibrin deposition in the advanced stages of disease, corresponding to the accumulation of collagen, IL-10 expression, and myofibroblasts expressing alpha smooth muscle actin (SMA). Together with the growing evidence that pulmonary microbleeds and coagulation play an active part in human pulmonary fibrosis, this data further supports our hypothesis that PECAM-1 expression is necessary for vascular barrier function control and regulation of homeostasis specifically, in the pulmonary environment.

Preclinical murine models for the testing of antimicrobials against Mycobacterium abscessus pulmonary infections: Current practices and recommendations.

Mycobacterium abscessus, a rapidly growing nontuberculous mycobacterium, is increasingly recognized as an important pathogen of the human lung, disproportionally affecting people with cystic fibrosis (CF) and other susceptible individuals with non-CF bronchiectasis and compromised immune functions. M. abscessus infections are extremely difficult to treat due to intrinsic resistance to many antibiotics, including most anti-tuberculous drugs. Current standard-of-care chemotherapy is long, includes multiple oral and parenteral repurposed drugs, and is associated with significant toxicity. The development of more effective oral antibiotics to treat M. abscessus infections has thus emerged as a high priority. While murine models have proven instrumental in predicting the efficacy of therapeutic treatments for M. tuberculosis infections, the preclinical evaluation of drugs against M. abscessus infections has proven more challenging due to the difficulty of establishing a progressive, sustained, pulmonary infection with this pathogen in mice. To address this issue, a series of three workshops were hosted in 2023 by the Cystic Fibrosis Foundation (CFF) and the National Institute of Allergy and Infectious Diseases (NIAID) to review the current murine models of M. abscessus infections, discuss current challenges and identify priorities toward establishing validated and globally harmonized preclinical models. This paper summarizes the key points from these workshops. The hope is that the recommendations that emerged from this exercise will facilitate the implementation of informative murine models of therapeutic efficacy testing across laboratories, improve reproducibility from lab-to-lab and accelerate preclinical-to-clinical translation.

Impact of Methylthioxylose Substituents on the Biological Activities of Lipomannan and Lipoarabinomannan in Mycobacterium tuberculosis.

Two lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM), play various, albeit incompletely defined, roles in the interactions of mycobacteria with the host. Growing evidence points to the modification of LM and LAM with discrete covalent substituents as a strategy used by these bacteria to modulate their biological activities. One such substituent, originally identified in Mycobacterium tuberculosis (Mtb), is a 5-methylthio-d-xylose (MTX) sugar, which accounts for the antioxidative properties of LAM. The widespread distribution of this motif across Mtb isolates from several epidemiologically important lineages have stimulated interest in MTX-modified LAM as a biomarker of tuberculosis infection. Yet, several lines of evidence indicate that MTX may not be restricted to Mtb and that this motif may substitute more acceptors than originally thought. Using a highly specific monoclonal antibody to the MTX capping motif of Mtb LAM, we here show that MTX motifs not only substitute the mannoside caps of LAM but also the mannan core of LM in Mtb. MTX substituents were also found on the LM and LAM of pathogenic, slow-growing nontuberculous mycobacteria. The presence of MTX substituents on the LM and LAM from Mtb enhances the pro-apoptotic properties of both lipoglycans on LPS-stimulated THP-1 macrophages. A comparison of the cytokines and chemokines produced by resting and LPS-activated THP-1 cells upon exposure to MTX-proficient versus MTX-deficient LM further indicates that MTX substituents confer anti-inflammatory properties upon LM. These findings add to our understanding of the glycan-based strategies employed by slow-growing pathogenic mycobacteria to alter the host immune response to infection.

Spectinamide MBX-4888A exhibits favorable lesion and tissue distribution and promotes treatment shortening in advanced murine models of tuberculosis.

The spectinamides are novel, narrow-spectrum semisynthetic analogs of spectinomycin, modified to avoid intrinsic efflux by Mycobacterium tuberculosis . Spectinamides, including lead MBX-4888A (Lee-1810), exhibit promising therapeutic profiles in mice, as single drugs and as partner agents with other anti-tuberculosis antibiotics including rifampin and/or pyrazinamide. To demonstrate that this translates to more effective cure, we first confirmed the role of rifampin, with or without pyrazinamide, as essential to achieve effective bactericidal responses and sterilizing cure in the current standard of care regimen in chronically infected C3HeB/FeJ mice compared to BALB/c mice. Thus, demonstrating added value in testing clinically relevant regimens in murine models of increasing pathologic complexity. Next we show that MBX-4888A, given by injection with the front-line standard of care regimen, is treatment shortening in multiple murine tuberculosis infection models. The positive treatment responses to MBX-4888A combination therapy in multiple mouse models including mice exhibiting advanced pulmonary disease can be attributed to favorable distribution in tissues and lesions, retention in caseum, along with favorable effects with rifampin and pyrazinamide under conditions achieved in necrotic lesions. This study also provides an additional data point regarding the safety and tolerability of spectinamide MBX-4888A in long-term murine efficacy studies.

Correlates of immune protection following cutaneous immunization with an attenuated Burkholderia pseudomallei vaccine.

Infections with the Gram-negative bacterium Burkholderia pseudomallei (melioidosis) are associated with high mortality, and there is currently no approved vaccine to prevent the development of melioidosis in humans. Infected patients also do not develop protective immunity to reinfection, and some individuals will develop chronic, subclinical infections with B. pseudomallei. At present, our understanding of what constitutes effective protective immunity against B. pseudomallei infection remains incomplete. Therefore, we conducted a study to elucidate immune correlates of vaccine-induced protective immunity against acute B. pseudomallei infection. BALB/c and C57BL/6 mice were immunized subcutaneously with a highly attenuated, Select Agent-excluded purM deletion mutant of B. pseudomallei (strain Bp82) and then subjected to intranasal challenge with virulent B. pseudomallei strain 1026b. Immunization with Bp82 generated significant protection from challenge with B. pseudomallei, and protection was associated with a significant reduction in bacterial burden in lungs, liver, and spleen of immunized mice. Humoral immunity was critically important for vaccine-induced protection, as mice lacking B cells were not protected by immunization and serum from Bp82-vaccinated mice could transfer partial protection to nonvaccinated animals. In contrast, vaccine-induced protective immunity was found to be independent of both CD4 and CD8 T cells. Tracking studies demonstrated uptake of the Bp82 vaccine strain predominately by neutrophils in vaccine-draining lymph nodes and by smaller numbers of dendritic cells (DC) and monocytes. We concluded that protection following cutaneous immunization with a live attenuated Burkholderia vaccine strain was dependent primarily on generation of effective humoral immune responses.

Disposable Dosators Intended for Dry Powder Delivery to Mice.

Dry powder inhalers offer numerous advantages for delivering drugs to the lungs, including stable solid-state drug formulations, device portability, bolus metering and dosing, and a propellant-free dispersal mechanism. To develop pharmaceutical dry powder aerosol products, robust in vivo testing is essential. Typically, initial studies involve using a murine model for preliminary evaluation before conducting formal studies in larger animal species. However, a significant limitation in this approach is the lack of suitable device technology to accurately and reproducibly deliver dry powders to small animals, hindering such models' utility. To address these challenges, disposable syringe dosators were developed specifically for intrapulmonary delivery of dry powders in doses appropriate for mice. These dosators load and deliver a predetermined amount of powder obtained from a uniform bulk density powder bed. This discrete control is achieved by inserting a blunt needle to a fixed depth (tamping) into the powder bed, removing a fixed quantity each time. Notably, this dosing pattern has proven effective for a range of spray-dried powders. In experiments involving four different model spray-dried powders, the dosators demonstrated the ability to achieve doses within the range of 30 to 1100 µg. The achieved dose was influenced by factors such as the number of tamps, the size of the dosator needle, and the specific formulation used. One of the key benefits of these dosators is their ease of manufacturing, making them accessible and cost-effective for delivering dry powders to mice during initial proof-of-concept studies. The disposable nature of the dosators facilitates use in animal procedure rooms, where cleaning and refilling reusable systems and weighing materials is inconvenient. Thus, developing disposable syringe dosators has addressed a significant hurdle in murine dry powder delivery for proof-of-concept studies, enabling researchers to conduct more accurate and reproducible preliminary studies in small animal models for pulmonary drug delivery.

Spray dried tigecycline dry powder aerosols for the treatment of Nontuberculous mycobacterial pulmonary infections.

Nontuberculous mycobacterial (NTM) pulmonary infections are a global health concern and a significant contributor to lung disease. Systemic therapies of a cocktail of antibiotics administered over a long period often lead to adverse reactions and/or treatment failure. NTM pathogens, such as Mycobacterium abscessus (Mabs), are notoriously difficult to treat due to resistance to many traditional antibiotics. However, the antibiotic tigecycline has demonstrated efficacy in vitro and in vivo against Mabs strains varying in drug susceptibility. Tigecycline exhibits instability in aqueous medium, posing delivery challenges, and has caused severe adverse gastrointestinal effects following intravenous administration, requiring treatment discontinuation. To mitigate both of these concerns, inhalation therapies using dry powder aerosols are proposed as an alternative administration route and means of delivery. Tigecycline dry powder formulations were prepared, characterized, and optimized to develop a therapeutic aerosol with low moisture, high dispersibility, and a large fraction of particles in the respirable size range (1-5 µm). The addition of lactose, leucine, and phosphate buffer salts was investigated to achieve additional stability, dispersibility, and tolerability. Preliminary delivery of the dry powders to Mabs-infected mice for 30 min per day over 7 d demonstrated a 0.91-log (87.7%) decrease in lung bacterial burden.

Characterization of spectinamide 1599 efficacy against different mycobacterial phenotypes.

Spectinamides are a novel series of spectinomycin analogs being developed for the treatment of tuberculosis. The preclinical lead spectinamide 1599 is an antituberculosis drug that possesses robust in vivo efficacy, good pharmacokinetic properties, and excellent safety profiles in rodents. In individuals infected with Mycobacterium tuberculosis or Mycobacterium bovis, causative agents of tuberculosis, the host immune system is capable of restraining these mycobacteria within granulomatous lesions. The harsh microenvironmental conditions of these granuloma lead to phenotypic transformation of mycobacteria. Phenotypically transformed bacteria display suboptimal growth, or complete growth arrest and are frequently associated with drug tolerance. Here we quantified the effect of spectinamide 1599 on log-phase and phenotypically tolerant isoforms of Mycobacterium bovis BCG using various in vitro approaches as a first indicator of spectinamide 1599 activity against various mycobacterial isoforms. We also used the hollow fiber infection model to establish time-kill curves and deployed pharmacokinetic/pharmacodynamic modeling to characterize the activity differences of spectinamide 1599 towards the different phenotypic subpopulations. Our results indicate that spectinamide 1599 is more efficacious against log phase bacteria when compared to its activity against other phenotypically tolerant forms such as acid phase bacteria and hypoxic phase bacteria, a behavior similar to the established antituberculosis drug isoniazid.